Fluorescent labeling is a very powerful technique for locating and tracking tagged objects, with applications in areas ranging from molecular dynamics and combinatorial chemistry to security systems and inventory control. However, this technique has several limitations. The number of objects which can be tracked simultaneously is limited by the fluorescent wavelengths available from the tags. In addition, this technique cannot provide information about three dimensional orientation relationships among objects.
A complete description of a three dimensional (3D) system includes analysis of the orientation of its components with respect to each other. For example, the chemical interactions of proteins with each other depend intimately on the spatial relationships they have with each other in all three dimensions. Characterization of an interface requires not only knowledge of the chemistry of the materials at either side of that interface but also of their orientation with respect to the interface and to each other.
Some 3D information about individual molecules or other complexes is available through scanning near-field optical microscopy (SNOM). However, SNOM is a relatively invasive technique which, in addition, can only analyze one small region of a sample at a time. Thus, the technique is not useful for high-throughput screening. Because of the small field of view, only a small total area can be analyzed, resulting in sampling errors. As a result, if a sample exhibits large amounts of variation, SNOM may not reveal the full variability of the sample. While far field microscopy can probe larger areas, it can only provide two-dimensional information about the orientation of a molecule with respect to the plane of the sample under observation.